This invention relates to novel DNA sequences that encode novel variant forms of acetohydroxy acid synthase enzyme (hereinafter AHAS). The AHAS enzyme is a critical enzyme routinely produced in a variety of plants and a broad range of microorganisms. Normal AHAS function is inhibited by imidazolinone herbicides; however, new AHAS enzymes encoded by the mutant DNA sequences function normally catalytically even in the presence of imidazolinone herbicides and, therefore, confer herbicide resistance upon the plant or microorganism containing them.
The novel DNA sequences are derived from corn and have a substitution of an amino acid at position 621 of the normal AHAS sequence. This substitution in the AHAS gene sequence results in a fully functional enzyme, but renders the enzyme specifically resistant to inhibition by a variety of imidazolinone herbicides. The availability of these variant sequences provides a tool for transformation of different crop plants to imidazolinone herbicide resistance, as well as providing novel selectable markers for use in other types of genetic transformation experiments.
The use of herbicides in agriculture is now widespread. Although there are a large number of available compounds which effectively destroy weeds, not all herbicides are capable of selectively targeting the undersirable plants over crop plants, as well as being non-toxic to animals. Often, it is necessary to settle for compounds which are simply less toxic to crop plants than to weeds. In order to overcome this problem, development of herbicide resistant crop plants has become a major focus of agricultural research.
An important aspect of development of herbicide-resistance is an understanding of the herbicide target, and then manipulating the affected biochemical pathway in the crop plant so that the inhibitory effect is avoided while the plant retains normal biological function. One of the first discoveries of the biochemical mechanism of herbicides related to a series of structurally unrelated herbicide compounds, the imidazolinones, the sulfonylureas and the triazolopyrimidines. It is now known (Shaner et al. Plant Physiol. 76: 545-546,1984; U.S. Pat. No. 4,761,373) that each of these herbicides inhibits plant growth by interference with an essential enzyme required for plant growth, acetohydroxyacid synthase (AHAS; also referred to as acetolacetate synthase, or ALS). AHAS is required for the synthesis of the amino acids isoleucine, leucine and valine.
The AHAS enzyme is known to be present throughout higher plants, as well as being found in a variety of microorganisms, such as the yeast Saccharomyces cerevisiae, and the enteric bacteria, Escherichia coli and Salmonella typhimurium. The genetic basis for the production of normal AHAS in a number of these species has also been well characterized. For example, in both E. coli and S. typhimurium three isozymes of AHAS exist; two of these are sensitive to herbicides while a third is not. Each of these isozymes possesses one large and one small protein subunit; and map to the IlvIH, IlvGM and IlvBN operons. In yeast, the single AHAS isozyme has been mapped to the ILV2 locus. In each case, sensitive and resistant forms have been identified and sequences of the various alleles have been determined (Friden et. al., Nucl. Acid Res. 13: 3979-3993, 1985; Lawther et al., PNAS USA 78: 922-928, 1982; Squires et al., Nucl. Acids Res 811: 5299-5313, 1983; Wek et al; Nucl. Acids Res 13: 4011-4027, 1985; Falco and Dumas, Genetics 109, 21-35, 985; Falco et al, Nucl. Acids Res 13; 4011-4027, 1985).
In tobacco, AHAS function is encoded by two unlinked genes, SuRA and SuRB. There is substantial identity between the two genes, both at the nucleotide level and amino acid level in the mature protein, although the N-terminal, putative transit region differs more substantially (Lee et al, EMBO J. 7: 1241-1248, 1988). Arabidopsis, on the other hand, has a single AHAS gene, which has also been completely sequenced (Mazur et al., Plant Physiol. 85:1110-1117, 1987). Comparisons among sequences of the AHAS genes in higher plants indicates a high level of conservation of certain regions of the sequence; specifically, there are at least 10 regions of sequence conservation. It has previously been assumed that these conserved regions are critical to the function of the enzyme, and that retention of that function is dependent upon substantial sequence conservation.
It has been recently reported (U.S. Pat. No. 5,013,659) that mutants exhibiting herbicide resistance possess mutations in at least one amino acid in one or more of these conserved regions. In particular, substitution of certain amino acids for the wild type amino acid at these specific sites in the AHAS protein sequence have been shown to be tolerated, and indeed result in herbicide resistance of the plant possessing this mutation, while retaining catalytic function. The mutations described therein encode either cross resistance for imidazolinones and sulfonylureas or sulfonylurea-specific resistance, but no imidazolinone-specific mutations were disclosed. These mutations have been shown to occur at both the SuRA and SuRB loci in tobacco; similar mutations have been isolated in Arabidopsis and yeast.
Imidazolinone-specific resistance has been reported elsewhere in a number of plants. U.S. Pat. No. 4,761,373 generally described an altered AHAS as a basis of herbicide resistance in plants, and specifically disclosed certain imidazolinone resistant corn lines. Haughn et al. (Mol. Gen. Genet. 211:266-271, 1988) disclosed the occurrence of a similar phenotype in Arabidopsis. Sathasivan et al. (Nucl. Acid Res. 18:2188, 1990) identified the imidazolinone-specific resistance in Arabidopsis as being based on a mutation at position 653 in the normal AHAS sequence. In accordance with the present invention, a gene encoding imidazolinone-specific resistance in a monocot has now been isolated and determined to be associated with a single amino acid substitution in a wild-type monocot AHAS amino acid sequence.
The present invention provides novel nucleic acid sequences encoding functional monocot AHAS enzymes insensitive to imidazolinone herbicides. The sequences in question comprise a mutation in the codon encoding the amino acid serine at position 621 in the corn (maize) AHAS sequence, or in the corresponding position in other monocot sequences. Other monocots, such as wheat, are also known to exhibit imidazolinone specific mutations (e.g., ATCC Nos. 40994-97). In corn, the wild type sequence has a serine at this position. In a preferred embodiment, the substitution is asparagine for serine, but alternate substitutions for serine include aspartic acid, glutamic acid, glutamine and tryptophane. Although the claimed sequences are originally derived from monocots, the novel sequences are useful in methods for producing imidazolinone resistant cells in any type of plant, said methods comprising transforming a target plant cell with one or more of the altered sequences provided herein. Alternatively, mutagenesis is utilized to create mutants in plant cells or seeds containing a nucleic acid sequence encoding an imidazolinone insensitive AHAS. In the case of mutant plant cells isolated in tissue culture, plants which possess the imidazolinone resistant or insensitive trait are then regenerated. The invention thus also encompasses plant cells, bacterial cells, fungal cells, plant tissue cultures, adult plants, and plant seeds that possess a mutant nucleic acid sequence and which express functional imidazolinone resistant AHAS enzymes.
The availability of these novel herbicide resistant plants enables new methods of growing crop plants in the presence of imidazolinones. Instead of growing non-resistant plants, fields may be planted with the resistant plants produced by mutation or by transformation with the mutant sequences of the present invention, and the field routinely treated with imidazolinones, with no resulting damage to crop plants.
The mutant nucleic acids of the present invention also provide novel selectable markers for use in transformation experiments. The nucleic acid sequence encoding a resistant AHAS is linked to a second gene prior to transfer to a host cell, and the entire construct transformed into the host. Putative transformed cells are then grown in culture in the presence of inhibitory amounts of herbicide; surviving cells will have a high probability of having successfully acquired the second gene of interest. Alternately, the resistant AHAS gene can be cotransformed on an independent plasmid with the gene of interest, whereby about 50% of all transformants can be expected to have received both genes.
The following definitions should be understood to apply throughout the specification and claims. A xe2x80x9cfunctionalxe2x80x9d or xe2x80x9cnormalxe2x80x9d AHAS enzyme is one which is capable of catalyzing the first step in the pathway for synthesis of the essential amino acids isoleucine, leucine and valine. A xe2x80x9cwild-typexe2x80x9d AHAS sequence is a sequence present in an imidazolinone sensitive member of a given species. A xe2x80x9cresistantxe2x80x9d plant is one which produces a mutant but functional AHAS enzyme, and which is capable of reaching maturity when grown in the presence of normally inhibitory levels of imidazolinone. The term xe2x80x9cresistantxe2x80x9d, as used herein, is also intended to encompass xe2x80x9ctolerantxe2x80x9d plants, i.e., those plants which phenotypically evidence adverse, but not lethal, reactions to the imidazolinone.